The development and utility of new high-throughput methods based on efficient extraction materials for purification and desalting of complex samples is of utmost importance. The complexity of biofluids, especially blood serum containing non-volatile salts, hinders the use of analytical tools. In proteomics and other biological fields, pre-treatment of sample is one of the most important procedures to pre-concentrate low concentrated analytes and to remove suppressing compounds such as salts or detergents before interfacing with mass spectrometry (MS). Compounds, especially at very low concentrations, often get their mass spectrometry signal suppressed by salts and other impurities. Therefore, a prior purification and separation step of biological samples is valuable. Several methods have been reported for sample treatment prior to MS analysis. These include dialysis, ultrafiltration, size-exclusion, affinity purification and solid phase extraction (SPE). Ultrafiltration and size-exclusion spin columns do not provide a sufficient degree of desalting. Dialysis often works well, but is difficult and expensive to automate. SPE is one of the most popular sample preparation methods to handle such problems and is very often used with matrix-assisted laser desorption/ionization (MALDI) MS. SPE with reversed phase (C4, C8 or C18) functional groups is the common strategy to desalt and concentrate protein and peptide samples. Other SPE materials based on hydrophilic interactions such as immobilized metal-ion affinity chromatography (IMAC) or porous graphitic carbon (PGC) have been proposed for purifying those categories of peptides prior to MS. PGC columns are normally used to purify carbohydrates and glycopeptides, but have recently been shown as an alternative or supplement to traditional reversed phase (RP) chromatography for separation of small and hydrophilic peptides prior to MALDI MS analysis.
The analysis of phosphorylated proteins and peptides is highly demanded, since protein phosphorylation is known as the most common post-translational modification found in nearly all cellular processes. Three amino acids, serine, threonine as well as tyrosine are common phosphorylation sites. Enrichment of phosphorylated proteins and peptides is generally required before mass spectrometric analysis because the abundance of phosphorylated forms is frequently low. Among various strategies used in enrichment of phosphorylated species the recently described metal oxide affinity chromatography (MOAC) is considered to be an efficient method. Metal oxides such as titanium dioxide (TiO2) or zirconium dioxide (ZrO2) have been successfully applied to selectively retain phosphopeptides from complex biological mixtures.
U.S. Pat. No. 6,048,457 describes a method for casting-in-place composite and/or non-filled structures which are useful as sorptive or reactive media or for size-based separations. Any particular housing size or configuration can be used, and the inclusion of a large amount of adsorptive particles in polymer is achieved while still maintaining the membrane three dimensional structure. In a first preferred embodiment, the composite structures comprise particles entrapped within a porous polymeric substrate, and are cast in-place into a housing such as a pipette tip, thereby providing an effective platform for micromass handling. However, the casting polymer is very hydrophobic and limits the overall chemistry of the tips.
U.S. Pat. application. 2006/0,115,384 discloses a sorption pipette tip for the extraction of a sample, or analyte, from a sample matrix with a method of using the sorption pipette tip to perform the extraction. The pipette tip has a coating of a sorptive material. The orifice of the tip may have different configurations that enhance extraction.
Pat. No. WO 02/40131 reveals a device for small volume sample preparation using tubes columns, and sheets, such as capillaries or pipette tips, in which particles of a separation medium, such as particles of a chromatography material used for sample preparation, are directly embedded in the solid material composing the tubes or columns or sheets.
U.S. Pat. application 2006/0,201,881 describes SPE devices including a plurality of packed nominally aligned capillary-channeled polymeric fibers for use as stationary phase materials. A plurality of fibers are packed together in a casing so as to provide good flow characteristics through the fibers and high surface area contact between a sample and the fibers. Different polymer compositions of the fibers permit the “chemical tuning” of the extraction process. The fibers can be physically or chemically derivatized to target specific analytes for separation from a test sample. Use of the fibers allows a wide range of liquid flow rates with very low backpressures.
Hui-Ching Hsieh et al. describes a phosphopeptide-selective pipette tip in which titanium dioxide nanoparticles were embedded in monolithic structure photopolymerized from ethylene glycol dimethacrylate. The lowest detectable amount of phosphopeptide was estimated to be at the low femtomole level (Journal of Chromatography A, Volume 1165, Issues 1-2, 21 Sep. 2007, Pages 128-135).
Jue-Liang Hsu et al. described a method for the fabrication of disposable plastic microtips by photopolymerization. C18 reversed-phase (C18) and ion metal affinity chromatography (IMAC) beads were immobilized on a plastic pipette tip, made of polypropylene materials, by photo-initiated polymerization. The combination of IMAC tips and MALDI-MS allowed the identification of phosphopeptides based on the phosphatase assay as well as the post-source decay (Electrophoresis 2004, 25, 3840-3847).
There is a need for a new extraction pipette tip technology that can successfully be applied for desalting and/or isolating biomolecules.
In this invention, a pipette tip was fitted with a monolithic polymer structure based on organic monomers of highly porous structure. The structure was formed with a hollow center allowing the sample and other liquids to pass through the monolithic bed structure. The monolithic bed was doped with different nano-powders such as diamond, fullerenes and metal oxides (TiO2 and ZrO2) to add functionality to the monolith. The particles have the additional advantage of modifying the stable, pre-monolith suspension to form a stable structure in the pipette tip with a hollow center. The hollow monolith structure was stable so that the mixture can be polymerized to form the hollow monolith structure. The permeability of final polymerized extraction tips was enhanced by the hollow channel in the center of the monolithic bed.